1. Field of the Invention
This invention relates to methods for stabilizing human interferon and to compositions of stabilized human interferon for use as pharmaceuticals. Stabilized interferon compositions comprise alpha interferon, omega interferon or mixtures thereof, and stabilizing agents. The invention also relates to medical dressings for delivering stable interferon to affected areas of the body.
2. Description of the Background
Interferons are proteins having a variety of biological actions including antiviral, immunomodulating and antiproliferative effects. Cellular production of interferons may be stimulated by numerous agents, including viruses. Interferons protect animal tissues and cells against viral attack and are an important host defense mechanism. Interferon may be produced endogenously by numerous cell types such as leukocytes, fibroblasts and lymphocytes, and may also be produced in cell culture or recombinantly. In most cases, interferons provide better protection to tissues and cells of the kind from which they have been produced than to other types of tissues and cells, indicating that human-derived interferon should be more efficacious in treating human diseases than interferons from other species. There are several distinct types of interferons, generally classified as alpha, beta, gamma and omega interferons, and a large number of variants thereof.
The alpha and omega interferons are derived primarily from leukocytes. Cell-derived interferon such as leukocyte interferon is difficult to purify to homogeneity and, as a consequence, is most often used as a crude or partially purified preparation. Leukocyte interferon preparations contain two molecular populations (alpha and omega interferons) that are distinguishable physically, chemically and biologically. Alpha interferon comprises approximately 80% of leukocyte interferon activity, and omega interferon comprises about 20% of leukocyte interferon activity. The leukocyte interferon forms can be separated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) into slow- and fast-migrating components as described by W. E. Stewart II and J. Desmyter (J. Virol. 67:68-73, 1975). The larger leukocyte interferon forms have apparent molecular weights of about 21,000-25,000 Daltons, while the smaller leukocyte interferon forms have apparent molecular weights of about 15,000-18,000 Daltons. The larger (omega) interferon are glycosylated, whereas the smaller (alpha) interferon are not. General discussions of these and other interferons can be found in various texts and monographs including: The Interferon System (W. E. Stewart, II, Springer-Verlag, N.Y. 1979); and Interferon Therapy (World Health Organization Technical Reports Series 676, World Health Organization, Geneva 1982). The method of administering interferon is an important factor in the clinical application of this important therapeutic agent. Systemic administration of interferon by either intravenous, intramuscular or subcutaneous injection has been most frequently used with some success in treating disorders such as hairy cell leukemia and Acquired Immune Deficiency Syndrome (AIDS)--related Kaposi's sarcoma. Among the problems inherent in intravenous, intramuscular or subcutaneous administration is that interferon can come into contact with uninfected or nonmalignant cells, and may thereby cause unwanted side effects such as fever, malaise and myalgia.
Condyloma acuminata (genital warts), a widespread, sexually transmitted disease of major public health concern, is a benign condition caused by human papilloma viruses (HPV). The Centers for Disease Control reported that the incidence of condyloma acuminata in the United States was at 160,000 in 1966, 1,400,000 in 1986, and over 3,000,000 in 1991. If this logarithmic growth continues, the number of infected patients is expected to reach about ten million by the year 2000.
In a recent study, HPV has also been shown to be the cause a number of cervical malignancies (F. X. Bosch et al., Second International Workshop on the Epidemiology of Cervical Cancer and Human Papilloma Virus. Int. J. Cancer 52:171-73, 1992). During the 16 years from 1974 through 1989, there have been more than a dozen studies published on the efficacy of topical applications of interferon for the treatment of condyloma acuminata (D. Ikic et al., Third Annual International Congress for Interferon research, Miami, Fla. 1982). These studies all utilized fleshly prepared batches of interferon ointment which were directly applied to the afflicted areas. Collectively, these studies resulted in a cure for 57% of those patients treated.
In some cases it would be preferable to administer interferon directly to the affected tissues or organs. This may be accomplished by direct injection of interferon into the diseased site such as done in selected cases of condylomata acuminata (genital warts), involving the external surfaces of genital or perianal areas. Interferon can also be administered by local topical application directly to the diseased site, such as a viral-induced skin lesion. Topical interferon may be useful for treatment of conditions such as condylomata acuminata, cervical dysplasia, rectal cancer, basal cell carcinoma, penile cancer or any other disorder responsive to interferon, particularly those linked to papilloma virus infection. Topical application in these cases could reduce the unwanted side effects associated with intramuscular, intravenous or subcutaneous administration. Also, the patient could self-administer topical interferon without the direct assistance of a qualified health-care worker. A topical interferon product could also promote patient compliance by providing a more pleasant therapeutic experience for the patient than interferon treatment via injection.
The lack of interferon stability in solutions and other products has heretofore limited its utility. Interferon products having enhanced storage stability should facilitate more wide-spread use of this important therapeutic agent. Several factors are important in designing a useful topical interferon preparation. First, conventional topical interferon preparations are generally considered unstable, and therefore are considered to have a limited shelf-life. A topical interferon preparation should be stabilized to prevent degradation over time and upon shipping and handling. Second, because interferon generally has a higher molecular weight than the molecular weights of the therapeutic agents usually administered in topical preparations, interferon should be incorporated into a substance which sufficiently holds the high molecular weight interferon in suspension during packaging, shipping and application, and yet also be able to release the substance from the preparation in a reasonable length of time once it has been applied to the diseased site. Third, the preparation must not adversely affect the antiviral activity of interferon.
Interferon has a well documented instability in ointment formulations. In one study, interferon-alpha ointment lost 80% of its activity in a period of two weeks at 4.degree. C. (Moller et al., Third Annual International Congress for Interferon research, Miami, Fla. 1982). In another study, a similar ointment lost 50% of its activity during this same time period when tested at room temperature and lost 50% of its activity (even when it contained the nonionic and cationic agents of Asculai and Miller, i.e., Tween-80 and Benzalkonium-Chloride (L. F. Estis et al., U.S. Pat. No. 4,680,175) by six hours at 37.degree. C. In studies performed by myself, preparations of interferon-alpha ointment lost 30% of their activity after one month at 4.degree. C., and 100% of its activity after one month at room temperature.
Asculai (European Patent Application No. 0077063) and Miller (U.S. Pat. No. 4,957,734), are directed to pharmaceutical compositions containing antiviral surface active agents. The compounds specifically referred to by these inventors are the nonionic surface active agents, such as non-oxynol-9, Brij, and Triton-X-100 (FIG. 1-A), the anionic surface active agents, sodium alkyl sulfonate and sodium alkyl benzene sulfonate, and two cationic surface active agents, the quaternary amine detergents, cetyl pyridinium chloride and benzalkonium chloride (FIG. 1-B). None of these compounds are individually useful for the stabilization of interferon. Further, Miller uses nonionic, anionic and cationic surface active agents in combination with interferon, but claims no stabilization of interferon and discloses that quaternary ammonium detergents such as cetyl pyridinium chloride and benzalkonium chloride are preferable cationic agents.
Nonionic surface active agents do not stabilize interferon and other similar proteins (K. Hasegawa et al., U.S. Pat. No. 4,675,184; L. F. Estis U.S. Pat. No. 4,680,175). Estis et al. is directed to the use of protease inhibitors such as alpha-1-antitrypsin inhibitor, alpha-2 macroglobulin, soybean inhibitor, N-alpha-tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride, and N-alpha-tosylphenylalanine chloromethyl ketone to stabilize topical interferon preparations. Hasegawa et al. is directed to anionic surface active agents such as sodium alkyl sulfates stabilize betainterferon (fibroblast-derived).
Stewart et al. (U.S. Pat. No. 3,981,991) is directed to interferon that can be stabilized by treating the antiviral agent with a combination of: 1) guanidine-hydrochloride or urea; 2) mercaptoethanol or ethanethiol; and 3) an agent selected from the group consisting of sodium dodecylsulfate, sodium decylsulfate, sodium dodecylsulfonate, dodecylamine and decylamine. Stewart et al. caution that interferon cannot be stabilized with only one or two of the aforesaid three agents.